WO2023087555A1 - 一种具有广谱中和活性的新型冠状病毒重组蛋白及其制备方法 - Google Patents

一种具有广谱中和活性的新型冠状病毒重组蛋白及其制备方法 Download PDF

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WO2023087555A1
WO2023087555A1 PCT/CN2022/075692 CN2022075692W WO2023087555A1 WO 2023087555 A1 WO2023087555 A1 WO 2023087555A1 CN 2022075692 W CN2022075692 W CN 2022075692W WO 2023087555 A1 WO2023087555 A1 WO 2023087555A1
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protein
recombinant
recombinant protein
rbd
preparation
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French (fr)
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高孟
沈钱通
庄昉成
毛子安
朱赟
陆绍红
陈刚
李思奇
安彤
张柯欣
张月
徐凯
陈彦霖
张温阳
侯云龙
包佳源
毛江森
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浙江普康生物技术股份有限公司
杭州医学院
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Publication of WO2023087555A1 publication Critical patent/WO2023087555A1/zh

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

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  • This application relates to the field of molecular biology technology, in particular to a novel coronavirus recombinant protein with broad-spectrum neutralizing activity and a preparation method thereof.
  • the amino acid mutations in the RBD region include D614G, N501Y, E484Q/K, K417N/T, and L452R, and RBD is the one that binds to ACE2 (angiotensin converting enzyme 2) in SARS-CoV-2
  • ACE2 angiotensin converting enzyme 2
  • the region is also the region where various neutralizing antibodies competitively bind. Therefore, amino acid mutations in the RBD region are most likely to cause viral immune escape.
  • the World Health Organization divides important variants into variants of concern (full English name: variant of concern, English abbreviation: VOC) and concern variants (English full name: variant of interest, English abbreviation: VOI).
  • the mutant strains of concern include: Alpha, Beta, Gamma, Delta, and Lambda.
  • Alpha, Beta, Gamma, Delta, and Lambda are examples of the Delta new crown mutant strains.
  • the Delta new crown mutant strain has the characteristics of strong transmission, short incubation period of infection, strong pathogenicity, and rapid disease progression, and has gradually become the main epidemic strain in the world. This led to a rebound of the epidemic in many countries and regions. Lambda is currently prevalent in 29 countries and regions, showing strong infectivity and reduced vaccine neutralization ability.
  • the purpose of this application is to provide a method for preparing a novel coronavirus recombinant protein with broad-spectrum neutralizing activity.
  • a method for preparing a novel coronavirus recombinant protein with broad-spectrum neutralizing activity the novel coronavirus recombinant protein is a CRM-RBD recombinant protein, and the amino acid sequence of the CRM-RBD recombinant protein is shown in SEQ ID NO: 1 , the nucleotide sequence encoding the CRM-RBD recombinant protein is shown in SEQ ID NO: 2, using the nucleotide sequence to prepare recombinant nanoprotein particles through the following steps:
  • the fermentation broth is subjected to cell disruption and centrifugation to obtain a crude inclusion body extract
  • the renaturation solution contains 2 g/L-6 g/L glycine, 0.5 g/L-2 g/L ethylene glycol Disodium amine tetraacetate and 0.1 g/L-0.5 g/L Tween-80, the pH value of the refolding solution is 8.5-10;
  • the refolded protein is concentrated by ultrafiltration, and then separated and purified by a molecular sieve chromatography column to obtain recombinant nano protein particles.
  • the nucleotide sequence encoding the CRM-RBD recombinant protein is inserted into an Escherichia coli expression vector by molecular cloning technology to obtain a recombinant expression plasmid, and the Escherichia coli recombinant expression plasmid is obtained by transforming Escherichia coli by a heat activation method. bacteria.
  • the Escherichia coli recombinant expression bacteria are cultured to the logarithmic growth phase at a temperature of 30°C-37°C, and then induced with an inducer for 4 hours to 24 hours at a temperature of 25°C-37°C, Obtain the fermentation broth.
  • the cell disruption method is selected from one or more of ultrasonic disruption and high-pressure homogenization.
  • the crude inclusion body extract is denatured and dissolved using a denaturing buffer
  • the denaturing buffer is composed of an acid-base buffer and the denaturant
  • the pH value of the denaturing buffer is 6- 9.
  • the denaturant is selected from one of urea and guanidine hydrochloride.
  • the acid-base buffer is selected from one of phosphate buffer, carbonate buffer and glycine buffer.
  • the present application also provides a novel coronavirus recombinant protein with broad-spectrum neutralizing activity, which is prepared according to the above-mentioned preparation method, and the particle size of the recombinant nanoprotein particle is 10 nanometers to 20 nanometers.
  • the recombinant nanoprotein particles after the recombinant nanoprotein particles are compatible and mixed with the aluminum adjuvant, they can induce the body to produce neutralizing and protective antibodies against the prototype strain of the new coronavirus, the beta variant strain and the delta variant strain.
  • the recombinant nano-protein particles after the recombinant nano-protein particles are compatible and mixed with the aluminum adjuvant, they can induce the body to generate a specific cellular immune response against the new coronavirus.
  • the recombinant nanoprotein particle after the recombinant nanoprotein particle is mixed with the aluminum adjuvant, it can produce stronger humoral immunity and cellular immunity than the RBD protein alone.
  • This application constructs a novel coronavirus recombinant protein, and uses a specific preparation method to prepare recombinant protein nanoparticles, so that it has broad-spectrum neutralizing antigenic activity against the new coronavirus prototype strain, beta variant strain, and delta variant strain.
  • the new coronavirus recombinant protein CRM-RBD is formed by fusion design of diphtheria toxin avirulent mutant CRM197 protein with immune adjuvant effect and RBD antigen structural protein.
  • the amino acid sequence is shown in SEQ ID NO: 1, of which 1-193 It is the functional region of CRM197, the 194th-208th position is the fusion link structure of two proteins, and the 209th-461st position is the SARS-CoV-2 antigen structural protein (RBD).
  • RBD SARS-CoV-2 antigen structural protein
  • CRM-RBD recombinant protein is expressed in Escherichia coli, chromatographic column separation, purification, and renaturation, and finally a novel coronavirus recombinant protein with good immunogenicity and broad-spectrum neutralizing activity is prepared.
  • Fig. 1 is the purified electrophoresis profile of the CRM-RBD recombinant protein in Example 1; wherein, M: Protein Molecular Weight Marker; 1: Bacterial Liquid Before Induction; 2: Bacterial Liquid After Induction; 3: Broken Bacteria; 5: column chromatography sample loading; 6: column chromatography elution; 7: sample before refolding; 8: sample after renaturation; 9: sample loading by molecular sieve; 10: molecular sieve purification.
  • M Protein Molecular Weight Marker
  • 1 Bacterial Liquid Before Induction
  • 2 Bacterial Liquid After Induction
  • 3 Broken Bacteria
  • 5 column chromatography sample loading
  • 6 column chromatography elution
  • 7 sample before refolding
  • 8 sample after renaturation
  • 9 sample loading by molecular sieve
  • 10 molecular sieve purification.
  • Figure 2 is a particle size scan of the novel coronavirus recombinant protein stock solution with broad-spectrum neutralizing activity obtained in Example 1.
  • Figure 3 is a transmission electron micrograph of the novel coronavirus recombinant protein stock solution obtained in Example 1 with broad-spectrum neutralizing activity.
  • Fig. 4 is the detection result of the neutralizing antibody described in Example 2.
  • FIG. 5 is the detection result of specific cellular immunity described in Example 3.
  • FIG. 6 is the detection result of the neutralizing antibody described in Example 4.
  • FIG. 7 is the detection result of specific cellular immunity described in Example 4.
  • Fig. 8 is the detection result of the neutralizing antibody described in Example 5.
  • FIG. 9 is the detection result of specific cellular immunity described in Example 5.
  • FIG. 10 is the test result of the antigen activity described in Example 6.
  • Recombinant Escherichia coli monoclonal colony pick the recombinant Escherichia coli monoclonal colony to 5 ml LB liquid medium for overnight culture at 37°C to obtain the recombinant Escherichia coli activation solution.
  • step (2) Take the recombinant Escherichia coli activation solution obtained in step (1), inoculate it into 5 ml of LB liquid medium at a volume ratio of 1:100, and culture it overnight at 37°C. Inoculate the bacteria liquid after overnight culture into fresh LB liquid medium at a ratio of 1:100 by volume, and cultivate the bacteria at 37 °C. : Optical Density) was 0.6, adding 1 mmol/L isopropyl- ⁇ -D-thiogalactoside, and induced expression at 30°C for 4 hours.
  • step (3) Use a centrifuge at 6000 rpm for 20 minutes to collect the colony precipitate after induction of expression in step (2), and resuspend the resulting colony precipitate with a bacteriostasis buffer at a mass volume ratio of 1:20, and use an ultrasonic instrument The cell wall of the colony was ultrasonically disrupted, and the sonicated bacterial liquid was centrifuged at 12,000 rpm for 30 minutes to collect the broken bacterial precipitate.
  • step (4) Place the recombinant protein purified solution obtained in step (4) in a tangential flow dialysis device containing a 10-kilodalton membrane bag and use a phase-changing solution for dialysis and phase-changing. After the protein solution after phase-changing is concentrated 8 times, Use a Superdex200pg molecular sieve chromatography column with a column volume of 300ml for purification.
  • the purification conditions are as follows: first equilibrate the chromatography column with phase-changing fluid, then load 15ml of sample into the chromatography column, then equilibrate the chromatography column with equilibrium fluid, and collect UV280
  • the protein solution corresponding to the second peak is the novel coronavirus recombinant protein stock solution with broad-spectrum neutralizing activity.
  • the purification results are shown in Figure 1.
  • step (6) Take the stock solution of novel coronavirus recombinant protein with broad-spectrum neutralizing activity obtained in step (5) and let it stand at room temperature for 10 minutes, then take 1 ml and use Zetasizer pro laser particle size analyzer in dynamic light scattering , the English abbreviation is: DLS) to measure the protein particle size, the result is shown in Figure 2, the average diameter of the obtained protein particles is 9.55 nanometers.
  • step (7) Take the novel coronavirus recombinant protein stock solution with broad-spectrum neutralization activity obtained in step (5) and add it dropwise on the copper mesh support membrane, drop 1% concentration of phosphotungstic acid negative staining solution, and stain for 1-2 minutes Use filter paper to absorb the negative dye solution, then wash the copper grid with deionized water for 1-2 times, then use filter paper to absorb the water, and observe it under a transmission electron microscope after drying. The results are shown in Fig. 3, under the electron microscope, evenly distributed protein particles with a diameter of about 10 nanometers can be seen.
  • the formula of the LB liquid medium 10 grams of tryptone, 5 grams of yeast extract, and 10 grams of sodium chloride, dissolved in water and adjusted to 1 liter.
  • the formula of the LB solid medium 10 grams of tryptone, 5 grams of yeast extract, 10 grams of sodium chloride, 15 grams of agar powder, and dilute to 1 liter with water.
  • Described antibacterial buffer formula 1.21 grams of Tris, 17.75 grams of sodium chloride, dissolved in water and settled to 1 liter, adjusted to 8.0 pH.
  • the formula of the denaturing liquid 1.21 grams of Tris, 480 grams of urea, dissolved in water to 1 liter, and adjusted to pH 7.0.
  • the formula of the eluent 1.21 grams of Tris, 58.44 grams of sodium chloride, 480 grams of urea, dissolved in water to 1 liter, and adjusted to pH 7.0.
  • phase-changing liquid 1.5 grams of glycine, 1.48 grams of disodium edetate, 0.2 grams of polysorbate 80, dissolved in water and adjusted to 1 liter, and adjusted to a pH value of 9.5.
  • CaptoS and Superdex200pg are commercially available fillers.
  • Example 2 Novel coronavirus recombinant protein with broad-spectrum neutralizing activity induces the body to produce broad-spectrum neutralizing antibodies
  • the novel coronavirus recombinant CRM-RBD protein with broad-spectrum neutralizing activity was prepared according to the preparation method of Example 1, and it was mixed and compatible with aluminum adjuvant to prepare a protein containing 50 ⁇ g/ml protein and 0.5 mg/ml aluminum adjuvant Vaccine samples.
  • mice serum was diluted (1:40-1:5120), mixed with the new coronavirus dilution (1000 half cell infection number (TCID50)/ml) by volume 1:1, and incubated at 37°C for 1 Hour.
  • TCID50 half cell infection number
  • step (3) Take the virus antibody mixture in step (3), add it to a 96-well cell culture plate covered with monolayer Vero E6 cells at 100uL/well, and add an equal volume of virus maintenance solution, each serum dilution Set up 8 multiple wells. At the same time, virus-free, serum-free and blank groups were set up as negative controls, cultured at 37° C. and 5% CO 2 (volume percentage) for 7 days, and the cell pathological changes were observed. Antibody serum titers were calculated by the Reed-Muench method.
  • Example 3 Novel coronavirus recombinant protein with broad-spectrum neutralizing activity induces broad-spectrum specific cellular immunity in the body
  • the novel coronavirus recombinant CRM-RBD protein with broad-spectrum neutralizing activity was prepared according to the preparation method of Example 1, and it was mixed and compatible with aluminum adjuvant to prepare a protein containing 50 ⁇ g/ml protein and 0.5 mg/ml aluminum adjuvant Vaccine samples.
  • Example 1 the novel coronavirus recombinant CRM-RBD protein with broad-spectrum neutralization activity was prepared, and at the same time, SEQ ID NO: 3 was inserted into the E. coli expression system to prepare the RBD protein according to the preparation method of Example 1.
  • Vaccine samples containing 50 micrograms/ml protein and 0.5 mg/ml aluminum adjuvant were prepared by mixing CRM-RBD protein and RBD protein with aluminum adjuvant respectively.
  • CRM-RBD group was immunized with 1 ml of CRM-RBD protein vaccine sample
  • RBD group was immunized with 1 ml of RBD protein vaccine sample
  • control group was immunized with 0.5 mg/ml aluminum adjuvant.
  • a total of three vaccinations were given with an interval of 28 days. Blood samples and spleens were collected two weeks after the last immunization.
  • Embodiment 5 Recombinant protein expression mode comparison
  • step (2) Place the purified recombinant protein solution obtained in step (1) in a tangential flow dialysis device containing a 10-kilodalton membrane bag and use phase-changing solution 1 and phase-changing fluid 2 to perform dialysis and phase-change respectively, and the After the protein solution was concentrated 8 times, it was purified with a Superdex 200pg molecular sieve chromatography column with a column volume of 300 ml to obtain a recombinant protein stock solution.
  • step (3) Take the phase-changing solution 1 and phase-changing solution 2 described in step (2) and obtain the recombinant protein stock solution obtained by dialysis and phase-changing at room temperature. Measure protein particle size. The results showed that the size of protein nanoparticles obtained by phase-changing solution 1 and phase-changing solution 2 was quite different. The average size of protein nanoparticles obtained by phase-changing solution 1 was 45 nanometers, and the average size of protein nanoparticles obtained by phase-changing solution 2 was 9.5 nanometers.
  • the resulting refolded protein was diluted to 0.1 mg/ml, and diluted with PBS diluent at a rate of 1:2-1:256; samples of each dilution were sequentially added to the wells of the enzyme plate pre-coated with ACE2 receptor protein , 100 microliters/well, add two wells for each diluted sample, and set up two negative wells in the other wells of the ELISA plate at the same time, and leave two wells as blank and zero wells, culture at 37°C for 0.5 hours, and wash the plate Add the enzyme-labeled antibody at a dilution of 4 times, incubate at 37°C for 0.5 hours, and wash the plate 4 times; place the chromogenic solution in a microplate reader at a wavelength of 450 nanometers, use a blank well to zero and measure the absorbance A value, and use it as a negative control 2.1 times the average A value of the well was used as the threshold value, and the maximum dilution factor greater than the threshold
  • phase-changing liquid formula used is as follows:
  • Phase-change solution 1 1.5 grams of glycine, 1.48 grams of disodium edetate, dissolved in water to 1 liter, and adjusted to pH 9.5.
  • Phase-change solution 2 1.5 grams of glycine, 1.48 grams of disodium edetate, 0.2 grams of polysorbate 80, dissolved in water to 1 liter, and adjusted to pH 9.5.
  • this application has constructed a new type of coronavirus recombinant protein CRM-RBD, and prepared recombinant protein nanoparticles by using a specific preparation method, so that it can simultaneously target the new coronavirus prototype strain, beta variant strain, and delta variant strain. broad-spectrum neutralizing antigenic activity.
  • the specific preparation method can effectively improve the immune activity of the protein and improve the body's humoral immunity and cellular immunity.

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Abstract

本申请提供了一种新型冠状病毒重组蛋白CRM-RBD及其制备方法，该重组蛋白能诱导机体产生广谱中和抗体。

Description

一种具有广谱中和活性的新型冠状病毒重组蛋白及其制备方法
相关申请
本申请要求2021年11月16日申请的，申请号为202111354553.3，发明名称为“一种具有广谱中和活性的新型冠状病毒重组蛋白及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及分子生物技术领域，特别是涉及一种具有广谱中和活性的新型冠状病毒重组蛋白及其制备方法。
背景技术
自严重急性呼吸系统综合征2型冠状病毒(英文全称为：severe acute respiratory syndrome coronavirus 2，英文简称为：SARS-CoV-2)在全球爆发以来，目前还没有针对SARS-CoV-2的特效药，因此能否研制出一款安全、有效的新冠疫苗成为预防和控制新冠肺炎的关键。目前全世界科研人员正在加速研发新冠疫苗，目前已有112种候选疫苗进入临床实验，有15种疫苗获得紧急使用，其中进入临床实验的亚单位疫苗有38种，2种亚单位疫苗获得紧急使用。
随着感染人数的增加和疫情的持续，新型冠状病毒不断进化和变异，陆续产生多种新冠病毒变异株。这些变异株的S蛋白发生氨基酸突变，特别是受体结合区或单克隆抗体结合位点氨基酸突变引起病毒的传播力和致病力改变以及部分免疫逃逸等。RBD区域(英文全称为：Receptor binding domain)的氨基酸突变有D614G、N501Y、E484Q/K、K417N/T、L452R，而RBD是SARS-CoV-2中与ACE2(血管紧张素转化酶2)结合的区域，也是多种中和 抗体竞争性结合的区域。因此，RBD区域的氨基酸突变最有可能导致病毒免疫逃逸。目前世界卫生组织将重要变异株划分为关切变异株(英文全称为：variant of concern，英文简称为：VOC)和关注变异株(英文全称为：variant of interest，英文简称为：VOI)。关切变异株有：Alpha、Beta、Gamma、Delta及Lambda，其中Delta新冠变异株具有传播力强、感染潜伏期短、致病性强、发病进程快等特点，逐渐成为全球主要的流行毒株，并导致多个国家和地区的疫情反弹。Lambda目前已在29个国家和地区流行，显示出较强的传染性和降低疫苗中和能力。
因此，有必要进一步研究针对新冠病毒不同变异株(具有交叉保护作用)且免疫效果较好的广谱或多价疫苗。
发明内容
基于此，本申请的目的是提供一种具有广谱中和活性的新型冠状病毒重组蛋白的制备方法。
一种具有广谱中和活性的新型冠状病毒重组蛋白的制备方法，所述新型冠状病毒重组蛋白为CRM-RBD重组蛋白，所述CRM-RBD重组蛋白的氨基酸序列如SEQ ID NO：1所示，编码所述CRM-RBD重组蛋白的核苷酸序列如SEQ ID NO：2所示，利用所述核苷酸序列通过以下步骤制备得到重组纳米蛋白颗粒：
将序列如SEQ ID NO：2所示的核苷酸序列插入表达载体并构建得到大肠杆菌重组表达菌；
培养所述大肠杆菌重组表达菌至对数生长期，用诱导剂诱导得到发酵液；
将所述发酵液通过细胞破碎和离心处理获得包涵体粗提物；
将所述包涵体粗提物用变性剂变性溶解，得到包涵体变性蛋白；
将所述包涵体变性蛋白用阳离子交换层析进行纯化，得到纯化重组蛋白；
用复性液对所述纯化重组蛋白进行液体置换复性，得到复性后蛋白，所述复性液含有2克/升-6克/升甘氨酸、0.5克/升-2克/升乙二胺四乙酸二钠和0.1克/升-0.5克/升吐温-80，复性液的pH值为8.5-10；
对所述复性后蛋白进行超滤浓缩，然后用分子筛层析柱进行分离纯化，得到重组纳米蛋白颗粒。
在其中一个实施例中，通过分子克隆技术将编码所述CRM-RBD重组蛋白的核苷酸序列插入大肠杆菌表达载体得到重组表达质粒，并通过热激活法转化大肠杆菌得到所述大肠杆菌重组表达菌。
在其中一个实施例中，所述大肠杆菌重组表达菌在30℃-37℃温度条件下培养至对数生长期，然后在25℃-37℃温度条件下用诱导剂诱导4小时-24小时，得到所述发酵液。
在其中一个实施例中，所述细胞破碎的方法选自于超声破碎和高压匀浆破碎中的一种或多种。
在其中一个实施例中，使用变性缓冲液将所述包涵体粗提物变性溶解，所述变性缓冲液由酸碱缓冲液和所述变性剂组成，所述变性缓冲液的pH值为6-9。
在其中一个实施例中，所述变性剂选自尿素和盐酸胍中的一种。
在其中一个实施例中，所述酸碱缓冲液选自磷酸盐缓冲液、碳酸盐缓冲液和甘氨酸缓冲液中的一种。
本申请还提供了一种具有广谱中和活性的新型冠状病毒重组蛋白，根据如上所述的制备方法制备得到，所述重组纳米蛋白颗粒的颗粒大小为10纳米-20纳米。
在其中一个实施例中，所述重组纳米蛋白颗粒与铝佐剂配伍混匀后，能诱导机体产生针对新冠病毒原型株、贝塔变异株和德尔塔变异株的中和保护抗体。
在其中一个实施例中，所述重组纳米蛋白颗粒与铝佐剂配伍混匀后，能诱导机体产生针对新冠病毒特异性的细胞免疫反应。
在其中一个实施例中，所述重组纳米蛋白颗粒与铝佐剂配伍混匀后，能 产生比单独RBD蛋白更强的体液免疫和细胞免疫。
本申请构建了一种新型冠状病毒重组蛋白，并采用特定的制备方法制备得到重组蛋白纳米颗粒，从而具有同时针对新冠病毒原型株、贝塔变异株、德尔塔变异株的广谱中和抗原活性。新型冠状病毒重组蛋白CRM-RBD通过具有免疫佐剂效果的白喉毒素无毒突变体CRM197蛋白与RBD抗原结构蛋白进行融合设计形成，氨基酸序列如SEQ ID NO：1所示，其中第1-193位为CRM197的功能区，第194-208位为两个蛋白的融合链接结构，第209-461位为SARS-CoV-2抗原结构蛋白(RBD)。CRM-RBD重组蛋白借助通过大肠杆菌表达后，采用特定的制备方法，可以有效提高蛋白的免疫活性，提高机体的体液免疫水平和细胞免疫水平。经过实验证明，将CRM-RBD重组蛋白通过大肠杆菌表达，层析柱分离、纯化、复性，最终制备得到具有良好免疫原性的具有广谱中和活性的新型冠状病毒重组蛋白。
附图说明
图1为实施例1中CRM-RBD重组蛋白的纯化电泳图谱；其中，M：蛋白分子量Marker；1：诱导前菌液；2：诱导后菌液；3：破菌；4：破菌液上清；5：柱层析上样；6：柱层析洗脱；7：复兴前样品；8：复性后样品；9：分子筛上样；10：分子筛纯化。
图2为实施例1中所得具有广谱中和活性的新型冠状病毒重组蛋白原液的粒径扫描图。
图3为实施例1中所得具有广谱中和活性的新型冠状病毒重组蛋白原液的透射电镜照片。
图4为实施例2中所述中和抗体检测结果。
图5为实施例3中所述特异性细胞免疫检测结果。
图6为实施例4中所述中和抗体检测结果。
图7为实施例4中所述特异性细胞免疫检测结果。
图8为实施例5中所述中和抗体检测结果。
图9为实施例5中所述特异性细胞免疫检测结果。
图10为实施例6中所述抗原活性检测结果。
具体实施方式
为了便于理解本申请，下面将对本申请进行更全面的描述，并给出了本申请的较佳实施例。但是，本申请可以以许多不同的形式来实现，并不限于本申请所描述的实施例。相反地，提供这些实施例的目的是使对本申请的公开内容的理解更加透彻全面。
除非另有定义，本申请所使用的所有的技术和科学术语与属于本申请的技术领域的技术人员通常理解的含义相同。本申请中在本申请的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本申请。本申请所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
实施例1 具有广谱中和活性新型冠状病毒重组蛋白的制备
(1)将核苷酸序列SEQ ID NO：2，通过Xho I和Nco I两个酶切位点将所合成的基因插入pET28a大肠杆菌表达质粒，得到重组表达质粒。将所得重组表达质粒通过42℃热激活法转入大肠杆菌BL21(DE3)感受态细胞中，并通过LB(英文全称为：Luria-Bertani)固体培养基平皿进行单克隆筛选，得到表达目的蛋白的重组大肠杆菌单克隆菌落，挑取重组大肠杆菌单克隆菌落至5毫升LB液体培养基37℃过夜培养，即得重组大肠杆菌活化液。
(2)取步骤(1)所得重组大肠杆菌活化液，按体积比1：100的比例接种于5毫升LB液体培养基，在37℃条件下过夜培养。将过夜培养后的菌液按体积比1：100的比例接种于新鲜的LB液体培养基中，37℃培养菌体，当菌体的OD 600值(600纳米处的吸光度值，OD的英文全称：Optical Density)为0.6时，加入1毫摩尔/升异丙基-β-D-硫代半乳糖苷，在30℃条件下诱导表达4小时。
(3)用离心机在6000转/分钟条件下离心20分钟收集步骤(2)诱导表 达后的菌落沉淀，所得菌落沉淀用破菌缓冲液以1：20的质量体积比重悬，并用超音仪超声破碎菌落细胞壁，超声破碎后的菌液用离心机12000转/分钟离心30分钟收集破菌沉淀。
(4)步骤(3)所得破菌沉淀用变性液溶解，12000转/分钟离心30分钟收集上清。所得上清液用5毫升柱体积(Column Volume,CV)的CaptoS阳离子层析柱进行纯化，层析条件如下：先用5个CV的变性液平衡层析柱，然后上样至层析柱中，再用5个CV的变性液平衡层析柱，最后用阶梯洗脱的方式洗脱得到重组蛋白纯化液，纯化结果如图1所示。
(5)将步骤(4)所得重组蛋白纯化液置于含有10千道尔顿膜包的切向流透析装置中用换相液进行透析换相，换相后的蛋白溶液浓缩8倍后，用300毫升柱体积的Superdex200pg分子筛层析柱进行纯化，纯化条件如下：先用换相液平衡层析柱，然后上样15毫升至层析柱中，再用平衡液平衡层析柱，收取UV280第二个峰所对应的蛋白溶液即得具有广谱中和活性新型冠状病毒重组蛋白原液。纯化结果如图1所示。
(6)取步骤(5)所得具有广谱中和活性新型冠状病毒重组蛋白原液室温静置平衡10分钟后，取1毫升用Zetasizer pro激光粒度仪在动态光散射(英文全称为：Dynamic Light Scattering，英文简称为：DLS)下测量蛋白粒径大小，结果如图2所示，所得蛋白颗粒平均直径为9.55纳米。
(7)取步骤(5)所得具有广谱中和活性新型冠状病毒重组蛋白原液滴加在铜网载膜上，滴上1％浓度的磷钨酸负染液，染色1分钟-2分钟后用滤纸吸去负染液，再用去离子水清洗铜网1-2次，再用滤纸吸去水，待干燥后置于透射电镜下观察。结果如图3所示，电镜下看以看到分布均匀的直径10纳米左右的蛋白颗粒。
所述LB液体培养基配方：胰化蛋白胨10克，酵母提取物5克，氯化钠10克，加水溶解定容至1升。
所述LB固体培养基配方：胰化蛋白胨10克，酵母提取物5克，氯化钠10克，琼脂粉15克，加水定容至1升。
所述破菌缓冲液配方：三羟甲基氨基甲烷1.21克，氯化钠17.75克，加 水溶解定容至1升，调节pH值至8.0。
所述变性液配方：三羟甲基氨基甲烷1.21克，尿素480克，加水溶解定容至1升，调节pH值至7.0。
所述洗脱液配方：三羟甲基氨基甲烷1.21克，氯化钠58.44克，尿素480克，加水溶解定容至1升，调节pH值至7.0。
所述换相液配方：甘氨酸1.5克，乙二胺四乙酸二钠1.48克，聚山梨酯0.2克80，加水溶解定容至1升，调节pH值至9.5。
所述CaptoS、Superdex200pg为市售商品化填料。
实施例2 具有广谱中和活性新型冠状病毒重组蛋白诱导机体产生广谱中和抗体
(1)按照实施例1的制备方法制备得到具有广谱中和活性新型冠状病毒重组CRM-RBD蛋白，与铝佐剂混合配伍制备得到含有50微克/毫升蛋白、0.5毫克/毫升铝佐剂的疫苗样品。
(2)取4周龄Babl/c小鼠，分成实验组及对照组，每组6只。实验组免疫1毫升疫苗样品，对照组免疫0.5毫克/毫升的铝佐剂。共免疫三针，免疫间隔28天。在末次免疫两周后采集血清。
(3)将小鼠血清倍比稀释(1:40-1:5120)，分别与新型冠状病毒稀释液(1000半数细胞感染数(TCID50)/毫升)按体积1:1混合，37℃孵育1小时。
(4)取步骤(3)中的病毒抗体混合液，按100uL/孔加至长满单层Vero E6细胞的96孔细胞培养板中，并补充等体积的病毒维持液，每个血清稀释度设置8个复孔。同时设立无病毒、无血清和空白组作为阴性对照，37℃、5％CO 2(体积百分比)条件下培养7天，观察细胞病变状况。按Reed-Muench两氏法计算抗体血清效价。
结果如图4所示：与对照组比，实验组小鼠血清具有明显的分别针对新冠病毒原型株、贝塔株及德尔塔株中和活性，且对新冠病毒原型株、贝塔株及德尔塔株的中和抗体水平差异不显著，表明所得新型冠状病毒重组蛋白具有广谱中和活性。
实施例3 具有广谱中和活性新型冠状病毒重组蛋白诱导机体产生广谱的 特异性细胞免疫
(1)按照实施例1的制备方法制备得到具有广谱中和活性新型冠状病毒重组CRM-RBD蛋白，与铝佐剂混合配伍制备得到含有50微克/毫升蛋白、0.5毫克/毫升铝佐剂的疫苗样品。
(2)取4周龄Babl/c小鼠，分成实验组及对照组，每组6只。实验组免疫1毫升疫苗样品，对照组免疫0.5毫克/毫升的铝佐剂。共免疫三针，免疫间隔28天。在末次免疫两周后采集血清。
(3)取Babl/c小鼠脾脏制备细胞悬液，用红细胞裂解液红细胞，PBS清洗两次，用10％FBS 1640培养基重悬，淋巴细胞计数。稀释至淋巴细胞数为5×10 6个/毫升。每孔加100微升细胞，分别用新冠病毒原型株、贝塔株及德尔塔株的RBD混合肽库作为刺激物，每只小鼠细胞作两个复孔，按照试剂盒说明书检测每只小鼠脾淋巴细胞中特异性分泌IFN-γ的效应T细胞数。
(4)结果如图5所示，所得新型冠状病毒重组亚单位蛋白疫苗可以有效诱导机体产生针对新冠病毒原型株、贝塔株及德尔塔株的特异性细胞免疫反应。
实施例4 融合抗原的比较
(1)按照实施例1的制备方法制备得到具有广谱中和活性新型冠状病毒重组CRM-RBD蛋白，同时将SEQ ID NO：3插入大肠杆菌表达系统按照实施例1的制备方法制备RBD蛋白。分别将CRM-RBD蛋白和RBD蛋白与铝佐剂混合配伍制备得到含有50微克/毫升蛋白、0.5毫克/毫升铝佐剂的疫苗样品。
(2)取4周龄Babl/c小鼠，分成CRM-RBD组、RBD组及对照组，每组6只。CRM-RBD组免疫1毫升CRM-RBD蛋白疫苗样品，RBD组免疫1毫升RBD蛋白疫苗样品，对照组免疫0.5毫克/毫升的铝佐剂。共免疫三针，免疫间隔28天。在末次免疫两周后采集血样和脾脏。
(3)将小鼠血清按1:5000稀释，分别加入等体积的1:1000稀释的不同变异株(包括原型株、贝塔株及德尔塔株)的辣根过氧化物酶标记的RBD蛋白混匀，37℃孵育25分钟，每孔加入100微升，37℃孵育15分钟，洗板5 次显色终止，用酶标仪在450纳米吸光度下读板值(OD 450)，同时以稀释液作为阴性对照。以血清抗体竞争性抑制RBD-HRP与ACE-2蛋白的亲和力作为来检测中和抗体活性，计算公式如下：抑制率＝(1-血清样品OD450÷阴性对照OD 450)×100％。结果显示：CRM-RBD组可以显著提升RBD蛋白的中和抗体水平(图6所示)。
(4)按照实施例3步骤(3)的方法，检测CRM-RBD组、RBD组及对照组小鼠的特异性细胞免疫反应。结果显示：CRM-RBD组可以显著提升RBD蛋白的特异性细胞免疫水平(图7所示)。
实施例5 重组蛋白表达方式比较
(1)将SEQ ID NO：3插入大肠杆菌表达系统按照实施例1的制备方法制备RBD蛋白，同时购买市售的CHO细胞表达的RBD蛋白(CHO-RBD)和293细胞表达的RBD蛋白(293-RBD)。分别将RBD蛋白、CHO-RBD蛋白以及293-RBD，分别与铝佐剂混合配伍制备得到含有50微克/毫升蛋白、0.5毫克/毫升铝佐剂的疫苗样品。
(2)取4周龄Babl/c小鼠，分成RBD蛋白组、CHO-RBD蛋白组、293-RBD组及对照组，每组6只。RBD蛋白组、CHO-RBD蛋白组、293-RBD组免疫1毫升蛋白疫苗样品，对照组免疫0.5毫克/毫升的铝佐剂。共免疫三针，免疫间隔28天。在末次免疫两周后采集血样和脾脏。
(3)按照实施例4步骤(3)的方法检测血清抗体的中和活性。结果如图8所示，RBD蛋白组、CHO-RBD蛋白组、293-RBD组均产生了较高的中和抗体。
(4)按照实施例3步骤(3)的方法检测小鼠的特异性细胞免疫反应，结果如图9显示，RBD蛋白组可以产生特异性细胞免疫反应，而CHO-RBD蛋白组、293-RBD组无法产生特异性细胞免疫反应。
实施例6 换相液配方筛选
(1)按照实施例1的制备方法制备得到重组蛋白纯化液.
(2)将步骤(1)所得重组蛋白纯化液置于含有10千道尔顿膜包的切向流透析装置中分别用换相液1和换相液2进行透析换相，换相后的蛋白溶液 浓缩8倍后，用300毫升柱体积的Superdex200pg分子筛层析柱进行纯化，得到重组蛋白原液。
(3)取步骤(2)所述换相液1和换相液2透析换相所得重组蛋白原液室温静置平衡10分钟后，取1毫升用Zetasizer pro激光粒度仪在动态光散射(DLS)下测量蛋白粒径大小。结果显示：换相液1和换相液2所得蛋白纳米粒大小差别较大，换相液1所得蛋白纳米颗粒平均大小为45纳米，换相液2所得蛋白纳米颗粒平均大小为9.5纳米。
(4)所得复性蛋白稀释至0.1毫克/毫升，用PBS稀释液按1:2-1:256进行倍比稀释；各稀释度样品依次加入预包被ACE2受体蛋白的酶标板孔中，100微升/孔，每个稀释样品加两孔，同时在酶标板的另外孔中设立阴性孔2个，并留2孔作空白调零孔，放入37℃培养0.5小时，洗板4次加入稀释度为酶标抗体，37℃培养0.5小时，洗板4次；显色液，置于酶标仪在450纳米波长下，用空白孔调零后检测吸光度A值，以阴性对照孔平均A值的2.1倍作为阈值，以大于阈值的最大稀释倍数作为蛋白抗原活性效价。结果显示：换相液1所得蛋白抗原活性为16，换相液2所得蛋白抗原活性为256。吐温80的加入明显提高了蛋白的抗原活性(图10)。
所用换相液配方如下：
换相液1：甘氨酸1.5克，乙二胺四乙酸二钠1.48克，加水溶解定容至1升，调节pH值至9.5。
换相液2：甘氨酸1.5克，乙二胺四乙酸二钠1.48克，0.2克聚山梨酯80，加水溶解定容至1升，调节pH值至9.5。
综上所述，本申请构建了一种新型冠状病毒重组蛋白CRM-RBD，并采用特定的制备方法制备得到重组蛋白纳米颗粒，从而具有同时针对新冠病毒原型株、贝塔变异株、德尔塔变异株的广谱中和抗原活性。CRM-RBD重组蛋白借助通过大肠杆菌表达后，采用特定的制备方法，可以有效提高蛋白的免疫活性，提高机体的体液免疫水平和细胞免疫水平。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未 对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本申请的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。因此，本申请专利的保护范围应以所附权利要求为准。

Claims (11)

  1. 一种具有广谱中和活性的新型冠状病毒重组蛋白的制备方法，其特征在于，所述新型冠状病毒重组蛋白为CRM-RBD重组蛋白，所述CRM-RBD重组蛋白的氨基酸序列如SEQ ID NO：1所示，编码所述CRM-RBD重组蛋白的核苷酸序列如SEQ ID NO：2所示，利用所述核苷酸序列通过以下步骤制备得到重组纳米蛋白颗粒：
    将序列如SEQ ID NO：2所示的核苷酸序列插入表达载体并构建得到大肠杆菌重组表达菌；
    培养所述大肠杆菌重组表达菌至对数生长期，用诱导剂诱导得到发酵液；
    将所述发酵液通过细胞破碎和离心处理获得包涵体粗提物；
    将所述包涵体粗提物用变性剂变性溶解，得到包涵体变性蛋白；
    将所述包涵体变性蛋白用阳离子交换层析进行纯化，得到纯化重组蛋白；
    用复性液对所述纯化重组蛋白进行液体置换复性，得到复性后蛋白，所述复性液含有2克/升-6克/升甘氨酸、0.5克/升-2克/升乙二胺四乙酸二钠和0.1克/升-0.5克/升吐温-80，所述复性液的pH值为8.5-10；
    对所述复性后蛋白进行超滤浓缩，然后用分子筛层析柱进行分离纯化，得到重组纳米蛋白颗粒。
  2. 根据权利要求1所述的制备方法，其中，通过分子克隆技术将编码所述CRM-RBD重组蛋白的核苷酸序列插入大肠杆菌表达载体得到重组表达质粒，并通过热激活法转化大肠杆菌得到所述大肠杆菌重组表达菌。
  3. 根据权利要求1所述的制备方法，其中，所述大肠杆菌重组表达菌在30℃-37℃温度条件下培养至对数生长期，然后在25℃-37℃温度条件下用诱导剂诱导4小时-24小时，得到所述发酵液。
  4. 根据权利要求1所述的制备方法，其中，所述细胞破碎的方法选自于超声破碎和高压匀浆破碎中的一种或多种。
  5. 根据权利要求1所述的制备方法，其中，使用变性缓冲液将所述包涵体粗提物变性溶解，所述变性缓冲液由酸碱缓冲液和所述变性剂组成，所述 变性缓冲液的pH值为6-9。
  6. 根据权利要求5所述的制备方法，其中，所述变性剂选自尿素和盐酸胍中的一种。
  7. 根据权利要求5所述的制备方法，其中，所述酸碱缓冲液选自磷酸盐缓冲液、碳酸盐缓冲液和甘氨酸缓冲液中的一种。
  8. 一种具有广谱中和活性的新型冠状病毒重组蛋白，其特征在于，根据权利要求1-7任一项所述的制备方法制备得到，所述重组纳米蛋白颗粒的颗粒大小为10纳米-20纳米。
  9. 根据权利要求8所述的新型冠状病毒重组蛋白，其中，所述重组纳米蛋白颗粒与铝佐剂配伍混匀后，能诱导机体产生针对新冠病毒原型株、贝塔变异株和德尔塔变异株的中和保护抗体。
  10. 根据权利要求8所述的新型冠状病毒重组蛋白，其中，所述重组纳米蛋白颗粒与铝佐剂配伍混匀后，能诱导机体产生针对新冠病毒特异性的细胞免疫反应。
  11. 根据权利要求8所述的新型冠状病毒重组蛋白，其中，所述重组纳米蛋白颗粒与铝佐剂配伍混匀后，能产生比单独RBD蛋白更强的体液免疫和细胞免疫。
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